Abstract Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated and promote hemolysis. Each year, 9000 new cases of AIHA are reported in the USA. Current therapies, including immunosuppression and/or splenectomy, have variable outcomes and are not effective in certain patients. Additionally, there is an 80% relapse rate in patients who have initial success with current therapies. Because autoantibodies are often against ubiquitous antigens, essentially all RBC units incompatible for many patients, thereby preventing transfusion of sufficient RBCs to treat anemia. Thus, the study of AIHA pathogenesis is significant due to our lack of mechanistic understanding and lack of efficacious therapies for some AIHA patients. Given the dangers of autoimmunity, the immune system has evolved multiple pathways of tolerance to self-antigens, including central and peripheral tolerance checkpoints. While AIHA occurs at a rate of 9000 new cases a year, up to 0.1% of asymptomatic blood donors have detectable RBC autoantibodies, demonstrating that tolerance to RBC antigens fails frequently. However, tolerance mechanisms with respect to RBC antigens are poorly understood, and represent an area of both clinical significance and also a source of important new basic understanding of immune tolerance. We have innovated a novel and tractable murine system to study immune tolerance to RBC antigens that allows testing of mechanistic hypotheses not possible in other systems. This model provides unique opportunities to 1) interrogate RBC-specific tolerance mechanisms, 2) evaluate potential prophylaxis therapies prior to onset of autoimmunity and 3) investigate potential therapeutics during active autoimmune disease. The proposed studies take advantage of these new tools and are designed to serve as a translational effort to elucidate how tolerance to RBC antigens is established and maintained, identify potential pathways that result in a breakdown of tolerance mechanisms, and provide insight into mechanisms of AIHA pathogenesis. An understanding of how the immune system responds to antigens on RBCs will provide significant insight into not only autoimmunity to RBCs, but immune responses to RBC antigens in general (thereby extending to inflammatory environments and exposure to allogeneic RBCs as a consequence of transfusions etc.), and serve as a rational basis for future human studies and potential development of novel therapies. As such, we propose the follow specific aims: Aim 1) Determine the subset(s) and phenotype of antigen presenting cells that participate in antigen presentation of RBC-derived antigens to lymphocytes, Aim 2) Determine how T cells are tolerized to self-derived RBC-specific antigens, and Aim 3) Test factors that can reverse tolerance to RBC autoantigens and play a role in AIHA pathogenesis.